Haemophilus influenzae is a major cause of disease worldwide. Six capsular serotypes (“a” to “f”) and an indeterminate number of acapsular (non-typeable) strains of H. influenzae are recognised. Type b capsular strains are associated with invasive diseases, including meningitis and pneumonia, while non-typeable H. influenzae (NTHi) is a primary cause of otitis media in children and respiratory tract infections in adults. Otitis media is a common childhood disease which accounts for the highest frequency of paediatric visits in the United States (Stool et al., Pediatr. Infect. Dis. Suppl., 8:S11-S14, 1989).
The development of a vaccine for NTHi diseases has proved difficult because of a lack of understanding of the antigens that confer protective immunity. Efforts in developing a NTHi vaccine have been focused on cell surface antigens such as outer membrane proteins and pili or fimbria (Kyd et al., Infect. Immun., 63:2931-2940, 1995; Deich et al., Vaccine Res., 2:31-39, 1995).
Recent advances in molecular genetics, molecular structure analysis and immunochemistry provide powerful tools which have permitted the identification of carbohydrate: structures as candidate vaccine antigens.
Gram-negative bacteria have an outer membrane comprised of components including proteins, lipoproteins, phospholipids, and glycolipids. The glycolipids comprise primarily endotoxin lipopolysaccharides (LPS). LPS are molecules comprised of a) a Lipid A portion which consists of a glucosamine disaccharide that is substituted with phosphate groups and long chain fatty acids in ester and amide linkages; b) a core polysaccharide which is attached to Lipid A by an eight carbon sugar, Kdo (ketodeoxyoctonate), and heptose, glucose, galactose, and N-acetylglucosamine; and, optionally, c) O-specific side chains comprised of repeating oligosaccharide units which, depending on the genera and. species of bacteria, may contain mannose, galactose, D-glucose, N-acetylgalactosamine, N-acetylglucosamine, L-rhamnose, and a dideoxyhexose (abequose, colitose, tyvelose, paratose, trehalose). LPS which lacks repeating O-side chains is sometimes referred to as short chain lipopolysaccharide, or as lipooligosaccharide (LOS). In this application, the term lipopolysaccharide (or LPS) includes short chain lipopolysaccharide and lipooligosaccharide (and LOS).
The major antigenic determinants of gram-negative bacteria are believed to reside in the complex carbohydrate structure of LPS. These carbohydrate structures vary significantly, even among different species of the same genus of gram-negative bacteria, primarily because of variations in one or more of the sugar composition, the sequence of oligosaccharides, the linkage between the monomeric units of the oligosaccharides and between the oligosaccharides themselves, and substitutions/modifications of the oligosaccharides (particularly the terminal oligosaccharide). For this reason, development of a vaccine having a broad spectrum effect against Haemophilus influenzae (particularly against NTHi) has been unsuccessful.
LPS is a bacterial component which has potential as a vaccine immunogen because of the antigenic determinants (“epitopes”) residing in its carbohydrate structures. However, the chemical nature of LPS detracts from its use in vaccine formulations; i.e., active immunization with LPS is unacceptable due to the inherent toxicity, in some animals, of the Lipid A portion. The pathophysiologic effects induced (directly or indirectly) by Lipid A of LPS in the bloodstream include fever, leucopenia, leucocytosis, the Shwartzman reaction, disseminated intravascular coagulation, abortion, and in larger doses, shock and death.
It has been established that vaccines comprised of capsular polysaccharides are effective at preventing human disease caused by the homologous encapsulated bacteria. These carbohydrate antigens are often poorly immunogenic in humans due to a lack of T-cell dependent response. However, by conjugating the specific polysaccharide antigen to a suitable protein carrier, the immunogenicity of the carbohydrate antigen can be greatly enhanced in patients who do not respond to the polysaccharide alone. Glycoconjugate vaccines based on the specific capsular polysaccharide of type b H. influenzae (Hib), e.g. ProHiBit™, and ActHib™, have already proven successful in the control of invasive Hib disease in infants. Capsular polysaccharide-protein conjugate Hib vaccines do not provide protection against disease caused by acapsular (non-typeable) strains of H. influenzae (i.e. against disease caused by NTHi) because they are only protective against infections caused by H. influenzae strains bearing the type b capsule.
Lipopolysaccharide (LPS) is a major NTHi cell surface antigen. LPS of Haemophilus influenzae has only been found to contain lipid A and oligosaccharide (OS) components. Because the lipid A component of LPS is toxic, it must be detoxified prior to conjugation to an immunogenic carrier, as discussed above.
Barenkamp et al. (Pediatr. Infect. Dis. J., 9:333-339, 1990) demonstrated that LOS stimulated the production of bactericidal antibodies directed against NTHi. McGehee et al. (Am. Journal Respir. Cell Biol., 1:201-210, 1989) showed that passive immunization of mice with monoclonal antibodies directed against LOS from NTHi enhanced the pulmonary clearance of NTHi.
Green et al. (Vaccines, 94:125-129, 1994) disclose a NTHi vaccine comprising a conjugate of NTHi oligosaccharide and the mutant nontoxic diphtheria protein CRM.sub.197. The lipid A moiety was removed from LOS by treatment with acid, followed by derivatizing the resulting OS with adipic acid dihydrazide (ADH) and coupling to CRM.sub.197. Despite the showing of Barenkamp et al. that LOS stimulated production of bactericidal antibodies against NTHi, the conjugates of Green et al. were determined to be poorly immunogenic after injection into mice. Moreover, the conjugates did not elicit bactericidal antibodies against NTHi.
Gu et al. (U.S. Pat. No. 6,207,157) is concerned with the detoxification of isolated NTHi LOS by removal of ester-linked fatty acids therefrom, so that it may be made suitable for vaccine preparation. However, Gu does not describe any other modifications to or desired chemical attributes of NTHi LOS.
There is currently no vaccine available to provide broad spectrum protection against infections caused by Haemophilus influenzae. Thus, there is a need for a vaccine having broad spectrum efficacy against Haemophilus influenzae, particularly NTHi.
In order to utilise an antigen for vaccine development, four essential criteria must be fulfilled. That is, the immunogenic epitope must be:                1. genetically stable;        2. conserved in all clinically relevant strains across the species;        3. accessible (in vitro and in vivo) to host immune mechanisms; and,        4. able to induce protective antibodies in vivo.        
There is a need to identify LPS carbohydrate epitopes of H. influenzae, particularly NTHi, which satisfy these criteria.